A method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol

Abstract

The present invention discloses a method for evaluating or screening efficacy substances for improving comfort after drinking alcoholic beverages and an alcoholic beverage. The method uses the enzyme activity of ALDH enzyme in AML12 cells after ethanol stimulation by natural active substances, the expression level of FGF21 protein in AML12 cells after ethanol stimulation, the level of reactive oxygen species in AML12 cells after ethanol stimulation, the enzyme activity of ALDH enzyme in C6 cells after ethanol stimulation, and the activation and proliferation of Con A-induced T lymphocytes after ethanol stimulation as indicators. The natural active substances screened by the present invention can more comprehensively and effectively improve the comfort after drinking white liquor.

Description

Technical Field

[0001] The present invention belongs to the technical field of food and health products, and particularly relates to a method for evaluating or screening functional substances for improving the comfort after drinking alcohol-containing beverages. Background Art

[0002] The comfort after drinking Chinese liquor has attracted much attention in recent years. Excessive drinking can lead to symptoms such as headache after drinking, thirst, physical incoordination, dizziness, and even coma. At the same time, organs such as the liver and brain will also undergo inflammation and oxidative damage. Therefore, some chemically synthesized anti-alcoholism drugs have emerged. For example, Metadoxine exerts its anti-alcoholism effect by promoting alcohol metabolism and protecting the liver from oxidative stress; Naloxone acts on the nervous system to promote wakefulness after drunkenness. However, these compounds have relatively large side effects and have limitations in improving the comfort after drinking Chinese liquor. Therefore, it is of great significance to develop a natural active substance with low toxicity and side effects and that can comprehensively improve the comfort after drinking Chinese liquor.

[0003] Forest plants are rich in various bioactive substances, such as flavonoids, polyphenols, saponins, terpenoids, polysaccharides, alkaloids, etc. A large number of studies have shown that these active substances are effective in relieving headache after drinking Chinese liquor through different pathways. For example, puerarin can significantly improve the antioxidant capacity of the liver in alcohol-drinking mice, and slow down apoptosis and inflammation. Dioscin can reduce alcoholic liver disease by reducing inflammatory cytokines and hepatic steatosis. Tea saponin can significantly reduce the absorption of ethanol and effectively protect liver tissue. Polysaccharides have multiple functions such as immune enhancement, anti-aging, and lipid-lowering, and are also high-quality raw materials for preparing functional health foods. It has been found that Dioscorea opposita polysaccharide can produce hepatoprotective effects through multiple pathways such as affecting ethanol metabolism and lipid peroxidation, and can significantly prolong the drunken tolerance time, shorten the sleep time, and reduce the mass concentrations of ethanol and acetaldehyde.

[0004] Currently, the screening of natural active substances for improving the comfort after drinking Chinese liquor from natural active substances mainly focuses on the effects of protecting the liver, antioxidant, anti-inflammatory, etc. after drinking. There are no relevant research results on screening active substances that do not cause headache after drinking, especially those that can relieve the symptoms of thirst after drunkenness. Therefore, it is very important to obtain excellent natural active substances that can not only protect the liver but also relieve headache, especially relieve dry mouth. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title, and such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] One object of the present invention is to provide a method for evaluating or screening functional substances that can improve the comfort after drinking alcohol, and the natural active substances obtained by screening can more comprehensively and effectively enhance the comfort after drinking Chinese liquor.

[0008] To solve the above technical problems, the present invention provides the following technical solution: A method for evaluating or screening functional substances that can improve the comfort after drinking alcohol, the method using the enzyme activity of aldehyde dehydrogenase in AML12 cells after ethanol stimulation by the functional substance, the protein expression level of fibroblast growth factor 21 in AML12 cells after ethanol stimulation, the reactive oxygen species level in AML12 cells after ethanol stimulation, the enzyme activity of catalase in AML12 cells after ethanol stimulation, the enzyme activity of aldehyde dehydrogenase in C6 cells after ethanol stimulation, the release amount of NO in macrophages induced by LPS after ethanol stimulation, and the activation and proliferation of T lymphocytes induced by Con A after ethanol stimulation as indicators.

[0009] As a preferred embodiment of the method for evaluating or screening functional substances that can improve the comfort after drinking alcohol of the present invention, it includes the steps of measuring the enzyme activity of aldehyde dehydrogenase in AML12 cells after ethanol stimulation by the functional substance, the protein expression level of fibroblast growth factor 21 in AML12 cells after ethanol stimulation, the reactive oxygen species level in AML12 cells after ethanol stimulation, the enzyme activity of aldehyde dehydrogenase in C6 cells after ethanol stimulation, and the activation and proliferation of T lymphocytes induced by Con A after ethanol stimulation;

[0010] When the enzyme activity of aldehyde dehydrogenase in AML12 cells after ethanol stimulation by the functional substance is higher than that of aldehyde dehydrogenase in AML12 cells after ethanol stimulation, and,

[0011] the protein expression level of fibroblast growth factor 21 in AML12 cells after ethanol stimulation by the functional substance is lower than that of fibroblast growth factor 21 in AML12 cells after ethanol stimulation, and,

[0012] the reactive oxygen species level in AML12 cells after ethanol stimulation by the functional substance is higher than that of reactive oxygen species in AML12 cells after ethanol stimulation, and,

[0013] the enzyme activity of aldehyde dehydrogenase in C6 cells after ethanol stimulation by the functional substance is lower than that of aldehyde dehydrogenase in C6 cells after ethanol stimulation, and,

[0014] when the activation and proliferation of T lymphocytes induced by Con A after ethanol stimulation by the functional substance is lower than that of T lymphocytes induced by Con A after ethanol stimulation, it is evaluated that the functional substance can improve the comfort after drinking alcohol.

[0015] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: statistical analysis is performed on the enzyme activity of aldehyde dehydrogenase in AML12 cells after ethanol stimulation by the efficacy substance, the protein expression level of fibroblast growth factor 21 in AML12 cells after ethanol stimulation, the reactive oxygen species level in AML12 cells after ethanol stimulation, the enzyme activity of catalase in AML12 cells after ethanol stimulation, the enzyme activity of aldehyde dehydrogenase in C6 cells after ethanol stimulation, the release amount of NO in LPS-induced macrophages after ethanol stimulation, and the activation and proliferation of Con A-induced T lymphocytes after ethanol stimulation. An efficacy substance with a comprehensive score higher than that of pure ethanol stimulation is evaluated as a preferred efficacy substance that can improve the comfort after drinking alcohol.

[0016] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: the performing of the statistical analysis includes,

[0017] Performing principal component analysis on the index data of various efficacy substances;

[0018] Using the principal component analysis method to calculate the scores of each principal component, and the calculation formula is:

[0019]

[0020] F i =W i1 X 1 +W i2 X 2 +…W in X n (2)

[0021] In the formula, W in is the coefficient in the linear combination corresponding to the nth index and the ith component, θ n represents the component matrix value corresponding to the nth index and the ith component, represents the square root of the eigenvalue of the ith component; X n represents the value of the nth index after data standardization; where, i = 1, 2, and n = 1, 2, 3, 4, 5, 6, 7;

[0022] Calculating the comprehensive score, and the calculation formula is:

[0023] F=α 1 F 1 +α 2 F 2 +…α i F i (3)

[0024] In the formula, α i represents the variance percentage of the ith principal component.

[0025] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: the preferred efficacy substances are oleuropein, hydroxytyrosol, maslinic acid, paeoniflorin, naringin, diosgenin, buddleoside, liquiritigenin, total flavonoids of Pueraria flos, polysaccharide of Hovenia dulcis, Gehua Decoction, curcumin, puerarin.

[0026] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: the concentration of oleuropein as an efficacy substance is 6.25 - 50 μM; the concentration of hydroxytyrosol as an efficacy substance is 12.5 - 100 μM; the concentration of maslinic acid as an efficacy substance is 6.25 - 50 μM; the concentration of paeoniflorin as an efficacy substance is 2.5 - 20 μM; the concentration of naringin as an efficacy substance is 12.5 - 100 μM; the concentration of diosgenin as an efficacy substance is 2.5 - 20 μM; the concentration of buddleoside as an efficacy substance is 12.5 - 100 μM; the concentration of liquiritigenin as an efficacy substance is 50 - 100 μM; the concentration of total flavonoids of Pueraria flos as an efficacy substance is 20 g / mL; the concentration of polysaccharide of Hovenia dulcis as an efficacy substance is 20 μg / mL; the concentration of Gehua Decoction as an efficacy substance is 20 μg / mL; the concentration of curcumin as an efficacy substance is 50 μM; the concentration of puerarin as an efficacy substance is 50 μM.

[0027] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: for the ethanol stimulation of the AML12 cells, the stimulation is carried out at an ethanol concentration of 2 - 5 v / v% for 4 h.

[0028] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: for the ethanol stimulation of the C6 cells, the stimulation is carried out at an ethanol concentration of 2 - 5 v / v% for 4 h.

[0029] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: for the macrophages induced by LPS after ethanol stimulation, at an ethanol concentration of 0.2 - 0.5 v / v%, the macrophages are induced with LPS for 48 h.

[0030] As a preferred embodiment of the method for evaluating or screening efficacy substances for improving the comfort after drinking alcohol in the present invention, wherein: for the T lymphocytes induced by Con A after ethanol stimulation, at an ethanol concentration of 0.2 - 0.5 v / v%, the T cells are stimulated with Con A for 48 h.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Through in vitro screening of a large number of natural active substances with the potential to improve the comfort after drinking Baijiu, the obtained oleuropein can not only effectively reduce the oxidative and inflammatory damage in the body caused by drinking Baijiu, but also effectively relieve symptoms such as "headache" and "dry mouth" after drinking Baijiu, thus more comprehensively and effectively improving the comfort after drinking Baijiu. At the same time, a feasible way and method for oleuropein to improve the comfort of Baijiu and realize its application are provided. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings. Among them:

[0034] Figure 1 Shows the effects of different Baijiu samples on the enzyme activity of aldehyde dehydrogenase (ALDH) in HepG2 and AML12 cells after ethanol stimulation;

[0035] Figure 2 Shows the effects of different Baijiu samples on the expression of CYP2E1 in hepatocytes after ethanol stimulation.

[0036] Figure 3 Shows the effects of different Baijiu samples on the enzyme activity of aldehyde dehydrogenase (ALDH) in C6 cells after ethanol stimulation.

[0037] Figure 4 Shows the effects of different Baijiu samples on the release of NO in microglial cell BV2 after ethanol stimulation.

[0038] Figure 5 Shows the effects of different Baijiu samples on the levels of reactive oxygen species (ROS) and the enzyme activity of catalase (CAT) in AML12 cells after ethanol stimulation.

[0039] Figure 6 Shows the effects of different Baijiu samples on the expression level of FGF21 protein in AML12 cells after ethanol stimulation.

[0040] Figure 7 Shows the effects of different Baijiu samples on the release of NO in LPS-induced mouse peritoneal macrophages after ethanol stimulation.

[0041] Figure 8 Shows the effects of different Baijiu samples on the activation and proliferation of Con A-induced mouse T lymphocytes after ethanol stimulation.

[0042] Figure 9 Shows the effects of 6 samples with hangover effects on the enzyme activity of ALDH in AML12 cells.

[0043] Figure 10 The ALDH enzyme activity in C6 cells by 6 samples with the effect of relieving alcohol intoxication.

[0044] Figure 11 The effect of 6 samples with the effect of relieving alcohol intoxication on the expression level of FGF21 protein in AML12 cells.

[0045] Figure 12 The effect of 6 samples with the effect of relieving alcohol intoxication on the level of reactive oxygen species (ROS) in AML12 cells.

[0046] Figure 13 The effect of 6 samples with the effect of relieving alcohol intoxication on the catalase (CAT) enzyme activity in AML12 cells.

[0047] Figure 14 The effect of 6 samples with the effect of relieving alcohol intoxication on the activation and proliferation of Con A-induced mouse T lymphocytes.

[0048] Figure 15 The effect of 6 samples with the effect of relieving alcohol intoxication on the NO release amount of LPS-induced mouse peritoneal macrophages.

[0049] Figure 16 The effect of 8 natural active substances on the aldehyde dehydrogenase (ALDH) enzyme activity in AML12 cells after ethanol stimulation.

[0050] Figure 17 The effect of 8 natural active substances on the aldehyde dehydrogenase (ALDH) enzyme activity in C6 cells after ethanol stimulation.

[0051] Figure 18 The effect of 8 natural active substances on the expression level of FGF21 protein in AML12 cells after ethanol stimulation.

[0052] Figure 19 The effect of 8 natural active substances on the level of reactive oxygen species (ROS) in AML12 cells after ethanol stimulation.

[0053] Figure 20 The effect of 8 natural active substances on the catalase (CAT) enzyme activity in AML12 cells after ethanol stimulation.

[0054] Figure 21 The effect of 8 natural active substances on the NO release amount of LPS-induced mouse peritoneal macrophages after ethanol stimulation.

[0055] Figure 22 The effect of 8 natural active substances on the activation and proliferation of Con A-induced mouse T lymphocytes after ethanol stimulation. Specific implementation manner

[0056] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description of the specific implementation manners of the present invention in conjunction with the embodiments of the specification.

[0057] In the following description, numerous specific details are set forth to facilitate a thorough understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0058] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that are mutually exclusive with other embodiments.

[0059] Unless otherwise specified, the raw materials used in the embodiments are all commercially purchased.

[0060] Example 1 Selection of Evaluation Indexes for Post - consumption Comfort of Baijiu at the Cellular Level and Optimization of Experimental Method Conditions

[0061] (1) Determination of the Effect of Wine Samples on the Activity of ALDH Enzyme in Specific Cells.

[0062] ① Determination of cell ALDH enzyme activity. HepG2 cell culture medium: 90% DMEM (Invitrogen), 10% fetal bovine serum (Gibco); AML12 cell culture medium: 89% DMEM / F - 12 (1:1) (Invitrogen), 10% fetal bovine serum (Gibco), 1% ITS Liquid Media Supplement (Sigma), 40 ng / ml Dexamethasone; The culture conditions are 37 °C and 5% CO 2 . Select cells in good condition, digest and count them, seed 5×10 6 cells (10 ml) into a 100 - mm cell culture dish, culture adherently for 40 h, change the medium. The alcohol control group changes to a medium with an ethanol content of 2 - 5% (v / v), and the sample group adds a wine sample with the same alcohol concentration, and continue to culture for 4 h. Collect the cells and determine the intracellular ALDH enzyme activity according to the instructions of the ALDH detection kit. A sample with high enzyme activity indicates that the sample can promote alcohol metabolism in the liver and reduce the damage of acetaldehyde to the liver.

[0063] ② Determination of C6 cell ALDH enzyme activity. C6 cell culture medium: 82.5% F - 12K Nutrient Mixture (Gibco), 15% horse serum, 2.5% high - quality fetal bovine serum. The culture conditions are 37 °C and 5% CO2 Select C6 cells in good condition, digest them and count the number of cells. Seed 4×10 6 cells (10 ml) into a 100-mm cell culture dish, and culture them adherently for 40 h. Change the medium. For the alcohol control group, change the medium to a medium with an ethanol content of 2-5% (v / v). For the sample group, add a wine sample with the same alcohol concentration, and continue the culture for 4 h. Collect the cells and measure the intracellular ALDH enzyme activity according to the instructions of the ALDH detection kit. A sample with low enzyme activity indicates that the sample can slow down the alcohol metabolism in the cerebellum and reduce the dizziness after drinking caused by the accumulation of acetic acid.

[0064] (2) Effect of the wine sample on the release of NO from lipopolysaccharide (LPS)-stimulated mouse microglial cells (BV2)

[0065] Evaluate the in vitro anti-inflammatory activity of the test sample by detecting the release amount of NO from mouse BV2 microglial cells. Seed the microglial cells at a density of 1×10 6 cells / well (2 mL) into a 6-well plate, and place it in a CO 2 incubator and culture adherently at 37 °C for 24 h, then change the medium. For the blank control group, add 2 mL of medium to each well. For the alcohol control group, add 1 mL of LPS (1 μg / mL) and 1 mL of medium with a final ethanol concentration of 0.2%-0.5% (v / v) to each well. For the test sample group, add 1 mL of LPS (1 μg / mL) and 1 mL of the wine sample with the same alcohol concentration to each well, and incubate for 24 h. Centrifuge the 6-well plate at 800 rcf / min for 5 min. Detect and analyze the effect of the sample on the release of NO from BV2 cells using the NO detection kit. The more significant the inhibitory effect on the release of NO, the better the inhibitory effect of the sample on the alcohol-related inflammatory response mediated by BV2 microglial cells.

[0066] (3) Effect of the wine sample on the indexes related to dry mouth after drinking in HepG2 / AML12 cells

[0067] Select cells in good condition, digest them and count the number of cells. Seed 5×10 6 cells (10 ml) into a 100-mm cell culture dish, and culture them adherently for 40 h. Change the medium. For the alcohol control group, change the medium to a medium with an ethanol content of 2-5% (v / v). For the sample group, add a wine sample with the same alcohol concentration, and continue the culture for 4 h. Collect the cells, lyse the proteins with WB and IP lysis buffer, then perform WB to detect the expression of FGF21 and analyze the gray scale of the band brightness with software. A sample with a low protein expression level of FGF21 indicates that the sample has the effect of relieving the symptom of thirst after drinking.

[0068] (4) Determination of the effect of the wine sample on the oxidation level of HepG2 / AML12 cells.

[0069] Select cells in good condition, and seed them at a density of 5×10 5Seed 6-well plates at a density of 1 cell / well (2 ml), culture adherently for 40 h, change the medium. For the alcohol control group, change to a medium with an ethanol content of 2-5% (v / v), and for the sample group, add wine samples with the same alcohol concentration, and continue to culture for 2 h. Collect the cells and detect the intracellular ROS level according to the ROS kit; detect the intracellular CAT level according to the CAT kit. Samples with a significantly increased intracellular ROS level indicate that the sample causes more severe oxidative damage to hepatocytes; samples with a significantly decreased intracellular CAT level indicate that the sample causes more severe oxidative damage to the liver.

[0070] (5) Effect of wine samples on the expression of CYP2E1 in hepatocytes after ethanol stimulation.

[0071] Experimental method: Extract the total RNA of hepatocytes after wine sample stimulation and use a protein and nucleic acid analyzer to measure the RNA concentration of the sample. Then, perform a reverse transcription reaction according to the RT-PCR kit instructions to obtain cDNA. Use CYP2E1 primers to perform PCR on the cDNA, take pictures in a nucleic acid imager, and perform gray-scale analysis on the CYP2E1 band to obtain the relative expression level of CYP2E1 mRNA.

[0072] (6) Effect of wine samples on the expression level of FGF21 protein in AML12 cells after ethanol stimulation. The experimental method is as follows: Seed AML12 cells (5×10 6 cells, 10 mL) in a 100 mm cell culture dish, culture adherently for 40 h, change the medium. For the alcohol control group, add 10 mL of 3% (v / v) alcohol for stimulation, and for the test sample group, add 10 mL of wine samples M3-1, M3-2, M3-3, M3-4, M3-5 with the same alcohol concentration, and continue to culture for 4 h. Collect the cells separately, lyse the proteins with WB and IP lysis buffer, and then perform WB to detect the expression of FGF21.

[0073] Example 2 Verification of the evaluation method for the comfort level after drinking of M3 series Baijiu at the cellular level

[0074] (1) Effect of test samples on the comfort level of Baijiu

[0075] According to the sensory evaluation of the Yanghe Distillery population, there is the following relationship between the comfort level after drinking M3 series Baijiu and the production year: The longer the year, the higher the comfort level. Five wine samples were respectively labeled as M3-1 (7 years), M3-2 (5 years), M3-3 (3 years), M3-4 (1 year), M3-5 (0 year) according to the production time, and a series of key indicators such as the ALDH enzyme activity, cell oxidation level, and inflammation level in specific cells after alcohol stimulation were detected for each sample according to the established evaluation method (Example 1). The results show that:

[0076] ① Effect of M3 series of Chinese Baijiu on the enzyme activity of aldehyde dehydrogenase (ALDH) in HepG2 and AML12 cells after ethanol stimulation

[0077] The results are as Figure 1 shown in a. In HepG2 cells, the better the comfort of the Chinese Baijiu, the higher the ALDH enzyme activity;

[0078] As Figure 1 shown in b. In AML12 cells, the better the comfort of the Chinese Baijiu, the higher the ALDH enzyme activity;

[0079] Overall, the order of ALDH enzyme activity in the Chinese Baijiu group from large to small is: M3-1 > M3-2 > M3-3 > M3-4 > M3-5. This indicates that the better the comfort of the Chinese Baijiu, the faster the alcohol metabolism in the liver.

[0080] ② Effect of M3 series of Chinese Baijiu on the expression of CYP2E1 in hepatocytes after ethanol stimulation

[0081] Figure 2 The results show that the effect of M3 series of Chinese Baijiu on the expression of CYP2E1 does not show an absolute correlation with comfort.

[0082] ③ Effect of M3 series of Chinese Baijiu on the enzyme activity of aldehyde dehydrogenase (ALDH) in C6 cells after ethanol stimulation

[0083] As Figure 3 shown, in C6 cells, the better the comfort of the Chinese Baijiu, the lower the ALDH enzyme activity. The order from small to large is: M3-1 < M3-4 < M3-2 < M3-3 < M3-5; this indicates that the longer the vintage and the better the comfort of the M3 series of Chinese Baijiu, the slower the alcohol metabolism in the cerebellum, reducing the dizziness after drinking caused by the accumulation of acetic acid.

[0084] ④ Effect of M3 series of Chinese Baijiu on the release of NO in microglial cell BV2 after ethanol stimulation

[0085] Figure 4 The results show that under the stimulation of alcohol, microglial cell BV2 will release a large amount of NO, leading to an increase in the inflammatory level of the brain tissue. The NO release caused by the Chinese Baijiu group is less than that of pure alcohol. The order from less to more is: M3-2 < M3-1 < M3-3 ≈ M3-5 < M3-4; this indicates that all M3 series of Chinese Baijiu can reduce the brain inflammation caused by alcohol, but there is no absolute correlation.

[0086] ⑤ Effect of M3 series of Chinese Baijiu on the levels of reactive oxygen species (ROS) and the enzyme activity of catalase (CAT) in AML12 cells after ethanol stimulation

[0087] As Figure 5 shown, the better the comfort of the Chinese Baijiu, the higher the CAT enzyme activity ( Figure 5 a) and the lower the ROS content in the cells (Figure 5 b) The lower the level, the order of oxidative damage in the liquor group from small to large is: M3-1 <M3-2<M3-3<M3-4<M3-5。5种不同年份的白酒均能不同程度地抑制酒精导致的胞内ROS水平升高,提高肝细胞内CAT水平,促进过氧化氢的分解。

[0088] ⑥ Such as Figure 6 As shown, compared with the alcohol group, the liquor group can significantly reduce the protein expression of FGF21. The more comfortable the liquor is, the lower the protein expression level of FGF21 is, and the better its effect in relieving the dry mouth symptoms after drinking may be. The order of the M3 series liquor in relieving the dry mouth symptoms after drinking is: M3-1>M3-2>M3-3>M3-4>M3-5.

[0089] ⑦Effects of M3 series liquor on NO release from mouse peritoneal macrophages induced by LPS after ethanol stimulation

[0090] like Figure 7 As shown, the M3 series liquor group can significantly inhibit the alcohol-related LPS-induced NO release of macrophages. The older the liquor, the lower the NO release, indicating that the older the liquor, the better the anti-inflammatory effect. The order of the liquor group's anti-macrophage-mediated inflammation level from large to small is: M3-1>M3-2>M3-3>M3-4>M3-5.

[0091] ⑧Effects of Yanghe M3 series liquors of different years on the activation and proliferation of mouse T lymphocytes induced by Con A after ethanol stimulation

[0092] like Figure 8 As shown, the M3 series of liquor can significantly inhibit the proliferation of alcohol-related Con A-activated T lymphocytes. The older the liquor, the higher the inhibition rate of T cell proliferation, indicating that the better the comfort level of the liquor, the better the anti-inflammatory effect. The order of the level of anti-T cell-mediated inflammation in the liquor group from large to small is: M3-1>M3-2>M3-3>M3-4>M3-5.

[0093] Example 3 Statistical analysis of various evaluation indicators of comfort after drinking M3 series liquor and calculation of comprehensive scores

[0094] The data of various evaluation indicators of the samples were statistically analyzed using SPSS software and the least squares method to obtain the comprehensive scores of the after-drinking comfort of the tested series of samples.

[0095] (1) Based on the results in Example 2, a comprehensive analysis was performed on five groups of different types of experimental data.

[0096] (2) PCA analysis was performed on the index data of 6 kinds of verification samples and isopentanol samples respectively, and 2 common factors were extracted, which can reflect more than 86% of the variance of the original variables.

[0097] (3) Use the principal component analysis method to calculate the scores of each principal component. The calculation formula is:

[0098] F i =W i1 X 1 +W i2 X 2 +…W in X n ①

[0099] In formula ①, represents the coefficient in the linear combination corresponding to the nth index and the ith component, and θ n represents the component matrix value corresponding to the nth index and the ith component, represents the square root of the eigenvalue of the ith component, and X n represents the value of the nth index after data standardization; i = 1, 2, n = 1, 2, 3, 4, 5, 6, 7.

[0100] (4) Calculate the comprehensive score. The calculation formula is:

[0101] F=α 1 F 1 +α 2 F 2 +…α n F n ②

[0102] In formula ②, α i represents the variance percentage of the ith principal component; i = 1, 2.

[0103] (5) Standardize the data of each evaluation index in Example 2 and substitute them into formulas ① and ② for statistical analysis. Among them, "Comprehensive Score 1" is the comprehensive score calculated according to all 9 indexes ( Figure 1 Two data in the index are taken as the ALDH enzyme activity in AML12 cells), and "Comprehensive Score 2" is the comprehensive score of 7 indexes excluding the CYP2E1 and BV2 cell NO indexes.

[0104] Table 1 Comprehensive Score of Post-drinking Comfort Evaluation of Samples

[0105]

[0106] The results are shown in Table 1. Regardless of whether 7 or 9 indicators are selected, the order of comprehensive scoring of the comfort after drinking liquor is M3-1>M3-2>M3-3>M3-4>M3-5. Therefore, the efficient comprehensive evaluation of the comfort after drinking liquor at the cellular level can select 7 indicators that are consistent with the market reputation and do not include CYP2E1 and BV2 cell NO indicators for preliminary screening and evaluation of the samples to be analyzed.

[0107] Example 4 Application of the method in Example 3 in evaluating the comfort level after drinking liquor using a recognized alcohol sobering substance

[0108] Six natural health factors or alcohol-relief products with high recognition (puerarin, curcumin, kudzu flower soup, kudzu flower flavonoids, Hovenia dulcis polysaccharides, and Neptune's Golden Bottle) were selected, and the key indicators of ALDH enzyme activity in specific cells after alcohol stimulation, FGF21 expression, cell oxidation level, and inflammation level were detected for each sample according to the established evaluation method (Example 1). Isoamyl alcohol, which is recognized to reduce the comfort of drinking liquor (based on the common concentration in liquor, 3 concentrations of 0.0065%, 0.0228%, and 0.08%) were set as controls to verify the reliability or effectiveness of the evaluation method established in Example 1. An alcohol control group was added, and only the same concentration of alcohol was added to the alcohol control group.

[0109] (1) Preparation of natural health factors or hangover products

[0110] Dissolve puerarin standard in DMSO to prepare 50mM puerarin stock solution. Dissolve curcumin standard in DMSO to prepare 50mM curcumin stock solution. According to the classic recipe of Gehua Decoction, weigh 20g each of Kudzu flower, Hovenia dulcis, Amomum villosum, and Cardamomum villosum, 6g each of Poria cocos, Tangerine peel, Codonopsis pilosula, Dry ginger, and Atractylodes macrocephala, soak in 1L water for 2h, boil for 1h, and then boil the residue in 800ml water for 1h, combine and rotary evaporate, and freeze-dry to obtain the sample of Kudzu flower decoction. Weigh 5g of Kudzu flower pollen, extract with 60% ethanol reflux, purify by adsorption with D101 macroporous resin, rotary evaporate, and freeze-dry to obtain the sample of Kudzu flower flavonoids. Take 2g of Hovenia dulcis powder, extract with distilled water by ultrasonic extraction (60℃, 362w, 65min), deproteinize by Sevage method, purify by macroporous resin D101 column, and freeze-dry after rotary evaporation to obtain a Hovenia dulcis polysaccharide sample. Take Neptune Jinzun product (liquid) and freeze-dry to obtain Neptune Jinzun sample.

[0111] (2) Effect of test samples on the comfort level of liquor

[0112] The prepared puerarin, curcumin, Gehua decoction, Gehua flavonoids, Hovenia dulcis polysaccharide, Haiwang Jinzun samples and isopentanol were respectively detected for a series of key indicators such as the activity of ALDH enzyme in specific cells, the expression of FGF21, and the cellular oxidation level and inflammation level after alcohol stimulation according to the established evaluation method (Example 3). The results showed that:

[0113] ① Compared with the alcohol group, the 6 samples with hangover effects all had an enhancing effect on the activity of ALDH enzyme in AML12 cells, and the effects of 20 μg / ml Hovenia dulcis polysaccharide and 20 μg / ml Haiwang Jinzun were particularly significant ( Figure 9 a), while isopentanol was not conducive to the increase in ALDH enzyme activity after drinking ( Figure 9 b). This indicates that these 6 recognized substances with hangover effects can promote alcohol metabolism in the liver and reduce the damage of acetaldehyde to the liver; isopentanol did not show a promoting effect on alcohol metabolism in the liver.

[0114] ② Compared with the alcohol group, the 6 samples with hangover effects could all reduce the activity of ALDH enzyme in C6 cells, and the effect of 20 μg / ml Hovenia dulcis polysaccharide was the best ( Figure 10 a), and isopentanol could also reduce the activity of ALDH enzyme in C6 cells ( Figure 10 b). This indicates that these 6 samples with hangover effects can slow down alcohol metabolism in the cerebellum and reduce the dizziness after drinking caused by the accumulation of acetic acid.

[0115] ③ Compared with the alcohol group, 20 μg / ml Hovenia dulcis polysaccharide and 20 μg / ml Gehua decoction could significantly inhibit the protein expression of FGF21 ( Figure 11 a); isopentanol could not reduce the protein expression level of FGF21 in cells ( Figure 11 b). This indicates that Hovenia dulcis polysaccharide and Gehua decoction may have a better effect on relieving thirst after drinking.

[0116] ④ The 6 samples with hangover effects could all inhibit the increase in intracellular ROS level caused by alcohol to varying degrees, and the effects of 50 μM curcumin and 20 μg / ml Haiwang Jinzun were the best ( Figure 12 a); while isopentanol led to an increase in intracellular ROS level ( Figure 12 b). The 6 samples with hangover effects could all significantly increase the level of CAT in hepatocytes and promote the decomposition of hydrogen peroxide, and the effects of 50 μM curcumin, 50 μM puerarin and 20 μg / ml Haiwang Jinzun were the best ( Figure 13 a); while isopentanol would further increase the intracellular ROS level and reduce the CAT level ( Figure 13 b). This indicates that the 6 samples with hangover effects can effectively reduce the oxidative damage of the liver caused by alcohol, while isopentanol may further exacerbate the oxidative damage of the liver.

[0117] ⑤Six samples with anti-hangover effects can significantly inhibit the proliferation of Con A-activated T lymphocytes related to alcohol. Among them, 20 μg / ml of puerarin flavone and 20 μg / ml ofhovenia polysaccharide have the best effects( Figure 14 a), while isopentanol has no inhibitory effect( Figure 14 b). Six samples with anti-hangover effects can significantly inhibit the release of NO from LPS-induced macrophages related to alcohol. Among them, 50 μM of puerarin, 20 μg / ml of puerarin flavone, 20 μg / ml of pueraria soup, and 20 μg / ml of Haiwang Jinzun have the best effects( Figure 15 a), while isopentanol cannot inhibit the release of NO( Figure 15 b). It shows that these six samples with anti-hangover effects can effectively inhibit alcohol-related inflammatory reactions, while isopentanol has no such effect.

[0118] (3) Standardize the data of various evaluation indicators of the six recognized samples with anti-hangover effects and isopentanol used in this example, and then substitute them into Formulas ① and ② in Example 3 for statistical analysis.

[0119] Table 2 Comprehensive score of the evaluation of the comfort after drinking the samples

[0120]

[0121] The results are shown in Table 2. The comprehensive evaluation results of the comfort after drinking the six samples with anti-hangover effects are all better than that of pure alcohol. The scoring order is total puerarin flavone > hovenia polysaccharide > pueraria soup > curcumin > puerarin > Haiwang Jinzun.

[0122] Table 3 Comprehensive score of the evaluation of the comfort after drinking isopentanol

[0123]

[0124] *Note: Tables 2 and 3 are two batches of experiments. There are certain differences in the performance of the cells after alcohol stimulation in the two batches of experiments (caused by factors such as cell passage number and cell state). In the same batch of experiments, the ratio of each experimental group to the ctrl group has high repeatability.

[0125] However, isopentanol, which is generally recognized as unfavorable to the comfort after drinking in Baijiu (Table 3), has a lower comprehensive score than pure alcohol at several common concentrations in Baijiu. The statistical results further illustrate the reliability or effectiveness of the evaluation method in Example 3.

[0126] Example 5 Screening of natural active substances based on the evaluation method of the comfort after drinking alcohol in Example 1

[0127] On the basis of a large number of primary screenings, eight natural active substances with good effects on improving the comfort after drinking Baijiu, such as naringin (CAS: 10236-47-2), liquiritigenin (CAS: 578-86-9), buddleoside (CAS: 480-36-4), dioscin (CAS: 19057-60-4), maslinic acid (CAS: 4373-41-5), hydroxytyrosol (CAS: 10597-60-1), paeoniflorin (CAS: 23180-57-6), oleuropein (CAS: 32619-42-4), etc., were selected for further in vitro screening.

[0128] (1) Effects of eight natural active substances on the enzyme activity of aldehyde dehydrogenase (ALDH) in AML12 cells after ethanol stimulation

[0129] The results are as Figure 16 a, Figure 16 b shown. Compared with the alcohol group, maslinic acid, paeoniflorin, hydroxytyrosol, and oleuropein all significantly increased the ALDH enzyme activity in AML12 cells, effectively promoting alcohol metabolism in the liver and reducing the damage of acetaldehyde to the liver. Among them, oleuropein had the most significant effect, and the optimal concentration of oleuropein was 25 μM.

[0130] (2) Effects of eight natural active substances on the enzyme activity of aldehyde dehydrogenase (ALDH) in C6 cells after ethanol stimulation.

[0131] The experimental results are as Figure 17 a, Figure 17 b shown. Compared with the alcohol group, naringin, liquiritigenin, paeoniflorin, and oleuropein all had a certain inhibitory effect on the ALDH enzyme activity in C6 cells, effectively slowing down alcohol metabolism in the cerebellum and reducing the dizziness after drinking caused by the accumulation of acetic acid. Among them, 6.25 μM oleuropein had the most obvious effect.

[0132] (3) Effects of eight natural active substances on the protein expression level of FGF21 in AML12 cells after ethanol stimulation.

[0133] The experimental results are as Figure 18 a, Figure 18 b, Figure 18 c shown. Compared with the alcohol group, naringin, maslinic acid, hydroxytyrosol, oleuropein, and dioscin significantly reduced the protein expression of FGF21, with the effect of relieving the symptom of thirst after drinking. Among them, oleuropein had the most significant effect, and the optimal concentration of oleuropein was 6.25 μM.

[0134] (4) Effects of eight natural active substances on the level of reactive oxygen species (ROS) in AML12 cells after ethanol stimulation.

[0135] The experimental results are as Figure 19 a,Figure 19 As shown in Fig. b. Compared with the alcohol group, the intracellular ROS levels decreased to a certain extent after treatment with the eight natural active substances, indicating that these eight natural active substances could effectively alleviate the oxidative damage to the liver caused by alcohol. Among them, the most significant effect was observed at the concentration range of 25 - 50 μM of luteoloside.

[0136] (5) Effects of the eight natural active substances on the catalase (CAT) activity in AML12 cells after ethanol stimulation.

[0137] The experimental results are shown in Figure 20 Fig. Figure 20 a and

[0138] b. Compared with the alcohol group, the intracellular CAT activity increased to a certain extent after treatment with the eight natural active substances, indicating that these eight natural active substances could effectively alleviate the oxidative damage to the liver caused by alcohol. Among them, the most significant effect was observed at the concentration of 100 μM of liquiritigenin.

[0139] The experimental results are shown in Figure 21 Fig. Figure 21 a and

[0140] (6) Effects of the eight natural active substances on the NO release in LPS-induced mouse peritoneal macrophages after ethanol stimulation.

[0139] The experimental results are shown in Figure 21 Fig. Figure 21 a and

[0140] (7) Effects of the eight natural active substances on the activation and proliferation of Con A-induced mouse T lymphocytes after ethanol stimulation.

[0141] The results are shown in Figure 22 Fig. Figure 22 a and

[0142] (8) Statistical analysis of the data of various evaluation indexes of the samples was carried out by SPSS software and the least square method to obtain the comprehensive score of the post-drinking comfort of the series of samples to be tested.

[0143] Table 4 Comprehensive score of the post-drinking comfort evaluation of the samples of the eight natural active substances

[0144]

[0145]

[0146] The results are shown in Table 2. The comprehensive comfort scores of the eight natural active substance samples after drinking are in the order of oleuropein > hydroxytyrosol > maslinic acid > paeoniflorin > naringin > diosgenin > buddleoside > glycyrrhizic acid. Among them, oleuropein, hydroxytyrosol, maslinic acid, paeoniflorin, naringin, diosgenin, and buddleoside at each concentration can improve the comfort after drinking. However, the comprehensive evaluation results of liquiritigenin at 12.5 μM and 25 μM are lower than those of pure alcohol, and it has little effect on the comfort after drinking.

[0147] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for evaluating or screening efficacy substances for improving comfort after drinking alcohol, characterized in that: this method statistically analyzes the enzyme activity of aldehyde dehydrogenase in AML12 cells after ethanol stimulation by efficacy substances, the protein expression level of fibroblast growth factor 21 in AML12 cells after ethanol stimulation, the reactive oxygen species level in AML12 cells after ethanol stimulation, the enzyme activity of catalase in AML12 cells after ethanol stimulation, the enzyme activity of aldehyde dehydrogenase in C6 cells after ethanol stimulation, the release amount of NO in LPS-induced macrophages after ethanol stimulation, and the activation and proliferation of Con A-induced T lymphocytes after ethanol stimulation; the said statistical analysis includes, performing principal component analysis on the index data of various efficacy substances; using the principal component analysis method to calculate the scores of each principal component, and the calculation formula is: F i = W i1 X 1 + W i2 X 2 + … W in X n (2) where, W in is the coefficient in the linear combination corresponding to the i-th component of the n-th index, and θ n represents the component matrix value corresponding to the i-th component of the n-th index, represents the square root of the eigenvalue of the i-th component; X n represents the value of the n-th index after data standardization; where, i = 1, 2, and n = 1, 2, 3, 4, 5, 6, 7; calculating the comprehensive score, and the calculation formula is: F = α 1 F 1 + α 2 F 2 + …α i F i (3) where α i represents the variance percentage of the i-th principal component; the comprehensive score of oleuropein as an efficacy substance at a concentration of 6.25 - 50 μM is higher than the comprehensive score before drinking alcohol.

2. The method according to claim 1, characterized in that: for the ethanol stimulation of AML12 cells, it is stimulated at an ethanol concentration of 2 - 5 v / v% for 4 h.

3. The method according to claim 1, characterized in that: for the ethanol stimulation of C6 cells, it is stimulated at an ethanol concentration of 2 - 5 v / v% for 4 h.

4. The method according to claim 1, characterized in that: for the LPS-induced macrophages after ethanol stimulation, at an ethanol concentration of 0.2 - 0.5 v / v%, LPS is used to induce macrophages for 48 h.

5. The method according to claim 1, characterized in that: for the Con A-induced T lymphocytes after ethanol stimulation, at an ethanol concentration of 0.2 - 0.5 v / v%, Con A is used to stimulate T cells for 48 h.

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